Abstract
Cellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of theCjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show thatCjLPMO10A is needed byC. japonicusto obtain efficient growth on both purified chitin and crab shell particles.
Highlights
Needed by C. japonicus to obtain efficient growth on both purified chitin and crab shell particles
The cavity positioned in close proximity to the active site in all chitin-specific Lytic polysaccharide monooxygenases (LPMOs) with known structure is in CjLPMO10A filled by Arg-197, Asp-202, and Glu-205, similar to what is observed in cellulose-oxidizing ScLPMO10C (Fig. 4A)
The growth data largely corroborate the enzymology for the two LPMOs produced by C. japonicus, showing that CjLPMO10B is specific for cellulose, and CjLPMO10A is specific for chitin
Summary
PRSET B_cjlpmo10A forward primer pRSET B_cjlpmo10A reverse primer (full-length) pRSET B_cjlpmo10A reverse primer (catalytic domain) pNIC-CH_cjcbm forward primer pNIC-CH_cjcbm reverse primer pNIC-CH_cjcbm forward primer pNIC-CH_cjcbm reverse primer. The only study so far addressing LPMO function in an in vivo context reports that the cellulose-active LPMO10B of Cellvibrio japonicus (CjLPMO10B) is important for the ability of this bacterium to grow on cellulose [36]. To cellulose-active CjLPMO10B, this microbe produces an additional LPMO, CjLPMO10A, that comprises three domains, including a CBM5 likely to bind chitin and a domain with unknown function. In addition to unraveling the functionality of the first three-domain LPMO ever characterized at this level of detail, these studies revealed a novel CBM that binds with high affinity to crystalline chitin. This chitin-binding module is the first representative of the new CAZy CBM73 family
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